Heme proteins play vital roles in physiology and medicine. Our broad goal is to understand the mechanisms of intracellular heme delivery and insertion into cytosolic proteins, and how this process is regulated in mammalian cells. We found that nitric oxide (NO) blocks cellular heme insertion into a range of heme proteins (NO synthases, cytochrome P450's, hemoglobin, and catalase). Using this information, and inducible NO synthase (iNOS) as our model heme protein, we found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a key player in the process. Our initial studies suggest that NO inhibits cellular heme insertion into iNOS by promoting a specific S-nitrosation of GAPDH, thereby altering GAPDH properties that are independent of its enzyme activity. We hypothesize that GAPDH plays an essential role in intracellular heme delivery through a non-traditional function that is regulated by its S-nitrosation. We propose cellular, biochemical, and biophysical approaches to test three aspects of our general hypothesis: Aim 1. Does GAPDH play a general role in intracellular heme delivery & homeostasis? We will test this by determining if GAPDH is involved in heme insertion into four proteins (hemoglobin, cytochrome P450, constitutive NOS, and catalase), and if GAPDH is involved in heme delivery to heme oxygenase 1 & 2. Aim 2. Do cells control their heme insertion reactions by regulating their capacity for protein S-nitrosation? We will test this by: (i) up or down-regulating the expression level of known cell denitrosylase enzymes (Thioredoxin-1 and GSH-NO reductase) to alter buildup of cellular S-nitrosoproteins (protein-SNO) in response to NO, and then (ii) determining if these changes alter levels of total protein-SNO and SNO-GAPDH in cells, and shift the NO sensitivity of cellular heme insertion in predictable ways. Aim 3. How are the heme binding, heme transfer, and protein interaction properties of GAPDH related to cellular heme delivery? We will investigate this by: (i) Measuring the heme binding parameters of pure GAPDH proteins that differ in their ability to support heme insertion in cells (wild type, S-nitrosated, point mutants), (ii) Evaluating interaction of GAPDH proteins with apo-protein targets and their ability to transfer bound heme in an in-vitro reconstitution system, and (iii) Solving the structure of the GAPDH-heme complex by protein crystallography and spectroscopy. Together, our studies will discern novel roles for NO, protein S-nitrosation, and GAPDH in intracellular heme delivery and insertion into proteins. This will advance our understanding of a fundamental process at the cellular and molecular levels, and will set the stage to investigate how these facets impact human health and disease at the organ and whole animal level. PUBLIC HEALTH RELEVANCE: Heme proteins exist in our cells and have many essential roles in our bodies. Our study will fill a tremendous gap in knowledge regarding how heme proteins are made in our cells, and how this process is regulated by nitric oxide, a gas that is made naturally by our bodies as a control agent. Our findings will touch many aspects of human health and disease, including blood formation, drug response, anemia, and inflammation.